Functional vitamin D derivatives and a method for determining 25-hydroxy-vitamin D and 1α, dihydroxy-vitamin D

ABSTRACT

Vitamin D compounds of formula (I) with a label attached to a spacer group in the 3 position are disclosed.                    
     In the above formula (I), X is an optionally substituted hydrocarbon group with a length of 0.8-4.2 nm, optionally containing the heteroatoms S, O, N or P; Y is H or OH; A is a label capable of binding with high affinity to a protein; R is an optionally substituted hydrocarbon side chain of a D vitamin or a D vitamin metabolite. Also disclosed is the preparation of formula (I).

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/EP99/04418 which has an Internationalfiling date of Jun. 25, 1999, which designated the United States ofAmerica.

The invention relates to derivatives of 25-hydroxy vitamin D, asynthesis thereof, and a method of measuring 25-hydroxy vitamin D and1,25-dihydroxy vitamin D in samples.

The D-vitamins or calciferols arise from their provitamins through acleavage, catalysed by sunlight, of the B-ring in the sterane rings.Their most important representatives are vitamin D₃ (cholecalciferol)and vitamin D₂ (ergocalci-ferol), which differ slightly only in the sidechains, but which—so far as known—are similarly metabolised and haveidentical biological effects. Whereas provitamin D₂ must be taken inwith the food, the provitamin D₃ can be formed in the human organism. Sofar as not more specifically designated by means of indices, the termvitamin D comprehends in the following in general all vitamin D forms.Vitamin D formed in the skin or taken in with food is bound in theplasma by vitamin D binding or transport proteins (DBP), transported tothe liver and there metabolised to 25-hydroxy vitamin D (25-OH-D). Thevitamin D binding protein DBP is also known as Gc-globulin or groupspecific component (J. G. Haddad in J. Steriod Biochem. Molec. Biol.(1995) 53, 579-582). Over 95% of the 25-hydroxy vitamin D measurable inthe serum is as a rule 25-hydroxy vitamin D₃. 25-Hydroxy vitamin D₂ isonly found in greater proportions if the person is receiving medicationwith vitamin D₂ or, as is frequently the practice in the United States,foodstuffs are supplemented with vitamin D₂.

25-Hydroxy vitamin D is the prevailing vitamin D metabolite in the bloodcirculation and its concentration in the serum generally indicates thevitamin D status, i.e. the extent to which vitamin D is available to theorganism. If needed, 25-hydroxy vitamin D is metabolised in the kidneysto 1α,25-dihydroxy vitamin D, a hormone-like substance with greatbiological activity. The determination of 1α,25-dihydroxy vitamin Dindicates how much vitamin D is present in the activated form.

BACKGROUND OF THE INVENTION

The determination of 25-hydroxy vitamin D in a sample is preferablyeffected in accordance with the principle of competitive protein bindinganalysis, whereby on the basis of the displacement of radioactive25-hydroxy vitamin D from the binding sites of a vitamin D bindingprotein, the 25-hydroxy vitamin D present in the sample can bequantified. Also, over the last several years, radioimmunoassays using¹²⁵I-labelled vitamin D derivatives and antibodies for vitamin Dderivatives have established themselves in diagnosis. The data of thenormal level of 25-hydroxy vitamin D in serum vary depending on thelaboratory. It is, however, agreed that the concentration of 25-hydroxyvitamin D in the serum is as a rule greater than 5 ng/ml and smallerthan 80 ng/ml. The competitive protein binding analysis requires the useof a radioactive vitamin D derivative which must have the same proteinbinding characteristics as 25-hydroxy vitamin D. The same applies alsofor the competitive binding analysis for the biologically active1α,25-dihydroxy vitamin D and other vitamin D metabolites.

European patent specifications 0 312 360 and 0 363 211, and Tanabe etal. in J. Chem. Soc., Chem. Commun. 1989, 1220-1221 and J. Nutri. Sci.Vitaminol., 1991, 37, 139-147, disclose various ¹²⁵I-labelled hydroxy-and dihydroxy vitamin D derivatives and their use in binding studies.These derivatives suffer the disadvantages that they are problematic toproduce and are extremely labile. Light, radioactive rays, protons,hydrogen, enzymes, free radicals or the presence of iodine in free orbound form have great effect on the configuration and the bindingcharacteristics of the vitamin D derivatives to vitamin D bindingprotein DBP or specific antibodies. Above all, they can cause orcatalyse a rotation of the A-ring in the sterane system. The3β-hydroxy-group of the vitamin D molecule is thereby rotated into thepseudo-1α-position, so that 5,6-trans-vitamin D is obtained. Theso-called pseudo-1α-hydroxy-analogs of vitamin D may be metabolisedsimilarly to vitamin D, but they have a structure which is different insignificant points and are not bound or are significantly more poorlybound by vitamin D binding proteins such as for example DBP/Gc-Globulinor anti-vitamin D antibodies.

DESCRIPTION OF RELATED ART

The above-described re-arrangement is to be understood as an example.Other chemical reactions and re-arrangements also occur. The sameapplies for ³H- or ¹⁴C-labelled vitamin b derivatives. These vitamin Dderivatives are likewise not so stable that they permit a reliablebinding analysis. The radioactive marking additionally increases thecosts of storage, transport and disposal and is generallydisadvantageous for health and the environment. Further the half-life of¹²⁵iodine is relatively short. On the other hand, a competitive bindinganalysis with ³H- and ¹⁴C-labelled vitamin D derivatives requiresparticular scintillation counters and is more demanding in terms ofequipment, with largely the same problems.

Ray et al., in Biochemistry, 1991, 30, 4809-4813 disclose the couplingof vitamin D₃ with various colouring groups. The detection sensitivityfor dye-labelled vitamin D₃ derivatives is, however, too small that onemight use them in a competitive binding analysis for natural vitamin Dmetabolites, apart from the fact that the dye-labelled derivatives arenot stable in serum and further are particularly light-sensitive.

BRIEF SUMMARY OF THE INVENTION

It is the object of the invention to make available vitamin Dderivatives which can be employed in a competitive binding analysis orquite generally in immunoassays of vitamin D metabolites such as25-hydroxy vitamin D and 1,25-dihydroxy vitamin D. This presumes thefollowing properties: first, that for the vitamin D derivatives, adetection sensitivity exists which is higher than, or lies in a lowerrange of concentrations than, the concentration of the sought aftervitamin D metabolites in the samples; second, that the derivatives arestable in serum, plasma or urine under the usual protonic conditions andare stable with the respect to serum enzymes; and finally, third, thatthe derivatives are sufficiently stable with regard to light andstorage, over weeks and months. This object is achieved by means ofvitamin D derivatives having the formula

wherein:

O represents the oxygen atom of an ether group;

X represents a substituted or non-substituted hydrocarbon group of 0.8to 4.2 nm length, preferably a C8- to C12-group, which may have theusual heteroatoms such as S, O, N or P, most particularly preferred anhexamido-, octamido- or decamido-amidopropylether linker group;

Y represents hydrogen or a hydroxy group;

A a functional group which is bound with high affinity by a bindingprotein such as an antibody or vitamin D binding protein DBP;

R the side group of a vitamin D metabolite, preferably the side group ofvitamin D₂ or D₃, particularly preferably the 25-hydroxylated side groupof vitamin D₂ or D₃.

A high affinity is present when the dissociation constant (K) betweenthe binding protein, e.g. the antibody or DBP, and the antigen or thefunctional group A is greater than 10⁸. A dissociation constant greaterthan 10¹⁶ is advantageous for many applications. In a preferredembodiment A is selected from biotin, digoxigenin, tyrosine, substitutedtyrosine, substituted amino acids, characteristic amino acid and peptidesequences, FITC, FITC-substituted tyrbsine, proteins and protein groupssuch as protein A and protein G or a further vitamin D derivative, mostparticularly preferred 25-hydroxy vitamin D.

The spacer group X is preferably selected from substituted andnon-substituted C-bodies having a length of 0.8 to 4.2 nm, preferablyabout 0.12 nm. Particularly preferred is an amino carboxylic acid, inparticular an amino undecanoic acid, peptide and keto group or asubstituted or non-substituted amino polyether radical having a lengthof 0.8 to 4.2 nm, preferably about 0.9 to 1.5 nm. This spacing betweenthe group A and the binding or detection site for the vitamin D radicalis necessary so that the binding proteins can bind to the binding siteconcerned in each case and thereby do not interfere with one another. Itis to be taken into consideration that for example for the vitamin Dbinding protein DBP (Gc-globulin) the 19-methylene group, if applicablethe 1-hydroxy group of the A-ring and the vitamin D side chain belong tothe recognition site and are received in a binding pocket. Similarapplies also for specific antibodies against the various vitamin Dderivatives. If the spacer group X is too short no further bindingprotein can bind to the selected functional group A along with thevitamin D binding protein. For the preferred example, this means thatwhen the functional biotin group is located within the binding pocket ofthe vitamin D binding protein it is no longer accessible for the secondbinding protein, for example the streptavidin. On the other hand, if thespacer group X is too long, molecular folding can arise which likewisehinders the simultaneous binding of two binding partners.

Further, the spacer group in accordance with the invention surprisinglyhas a steric effect, since it clearly actively hinders a 180° degreerotation of the A-ring. It is suspected, without being restricted tothis theory, that the 3β-oxygen atom of the ether group on the A-ring ishydrated corresponding to a natural hydroxy group and so prevents anattack on the 5,6-double bond, apart from other electronic and stericeffects. A further important aspect is that the ether group inaccordance with the invention cannot be dissociated by the esteraseswhich are always present in serum or plasma.

Most particularly preferred is 25-hydroxyvitamin-D₃-3β-3′[6-N-(biotinyl)hexamido]amidopropylether of the formulaII

and the 1α-hydroxy- and vitamin D₂ analogs.

Further preferred are derivatives which contain as the second functionalgroup a vitamin D radical. The advantage of these derivatives is thatthey contain no groups and compounds foreign to the system and so allowan increased sensitivity and reliability of the competitive bindinganalysis, also because they compensate, in a quantitative detection, forpossible binding peculiarities of first and second binding of thevitamin D binding protein. Particularly preferred are compounds of thefollowing formula III:

wherein R, Y and X are defined as in formula I above. Thereby,symmetrical vitamin D derivatives are particularly favourable.

The 25-hydroxy- and 1α,25-dihydroxy vitamin D derivatives in accordancewith the invention are surprisingly stable with respect to light,storage and serum and allow in all competitive immune diagnostic methodsa sensitive, reliable quantitative determination of vitamin Dmetabolites such as 25-hydroxy- and 1α,25-dihydroxy vitamin D, forexample for routine diagnostic use in human or veterinary medicine andin research.

In accordance with the invention the compound having formula I isobtained by means of a method including the steps: a) cyanoethylation ofthe 3-hydroxy group of vitamin D or 25-hydroxy vitamin D withacrylonitrile in a suitable solvent such as acetonitrile in the presenceof potassium hydride and tertiary butanol; b) reduction of the resultingnitrile group with a mixture of lithium hydrid and lithium aluminiumhydride to an amine; and c) linking a spacer group, if appropriate witha functional group A, to the amine, for example biotinylation of thecompound with an active biotinylation reagent such as LC-BHNS or, toobtain a vitamin D derivative in accordance with formula III, couplingof two amino-vitamin D groups, by means of condensation, with adicarboxylic acid such as sebacinic acid, by means of carbodiimide.

The method in accordance with the invention for the production offunctional vitamin D derivatives gives higher yields with shorterreaction times. Different from conventional methods, there is effectednamely in step a) the cyanoethylation of the 3-hydroxy group in thepresence of potassium hydride and tertiary butanol. By this means it isachieved that cyanoethylation is effected only at the 3-hydroxy group ofvitamin D and the other hydroxy groups of the vitamin D are protectedfrom reaction. The reaction is effected at 0 to 20° C., preferably at 5to 8° C. in a neutral solvent medium such as acetonitrile.

In the subsequent reduction, the nitrile group of the cyanoethylether isquantitatively reduced into the amine, which can then be relativelysimply linked with another functional group, for example by means ofreaction with a commercial available biotinylation reagent.

The invention includes additionally the use of the functional vitamin Dderivatives in accordance with the invention in methods for detecting25-hydroxy- and 1α,25-dihydroxy vitamin D in serum, plasma, urine oranother sample. Here, the functional vitamin D conjugate in accordancewith the invention is employed either as an intermediate, whereby thevitamin D binding protein and native vitamin D metabolites compete forthe binding site, or is employed itself as competitive binding componentto native vitamin D. The quantitative detection method is preferably anEIA, ELISA, RIA, IRMA, LiA or ILMA, FIA or IFMA in test systems whichare to be worked manually or in versions adapted to automatic testingmachines, in liquid phase as well as solid phase technology.

A particularly preferred method for detecting 25-hydroxy- and1α,25-dihydroxy vitamin D derivatives include the steps: a) coating acarrier with streptavidin, b) addition of one or a plurality of amultifunctional biotin-vitamin D derivatives, c) addition of the sampleand a defined quantity of vitamin D binding protein, d) detection of thebound binding protein with labelled anti-vitamin D binding proteinantibodies. The labelling of the anti-vitamin D binding proteinantibodies can be direct, for example a radioactive marking, or alsoindirect, for example by an enzyme or an active enzyme fragment such asperoxidase, which is capable of catalysing a colour reaction.

A further preferred method for detecting 25-hydroxy- and 1α,25-dihydroxyvitamin D derivatives includes the steps: a) coating a carrier withanti-vitamin D binding protein antibodies, b) adding the vitamin Dbinding protein, c) adding the sample and a defined quantity ofbiotin-vitamin D derivative, d) detecting the quantity of boundderivative with labelled streptavidin. The streptavidin is preferablyindirectly labelled with peroxidase; the carrier is preferably areaction vial wall, for example of a microtitration plate, or particlesof polymer or magnetic material or both, for example plastic orcellulose microparticles.

These methods make possible a non-radioactive quantitative detection of25-hydroxy- and 1,25-dihydroxy vitamin D, without extensive safetymeasures being required. The competitive methods proposed here are thussuitable for routine investigations within in the scope of osteoporosisprophylaxis, in the case of a suspected D-hypovitaminosis orD-hypervitaminosis, for diagnostics in general, and in research.

A further aspect of the invention is a kit for detecting vitamin Dmetabolites such as 25-hydroxy- and 1,25-dihydroxy vitamin D, whichinter alia contains the functional vitamin D derivative in accordancewith the invention. The kit includes a vitamin D binding protein(Gc-globulin) which can be freely selected, anti-vitamin D bindingprotein antibodies, streptavidin and pre-prepared or non-pre-preparedmicrotitration plates and/or magnetic or other microparticles and otherreagents.

BRIEF DESCRIPTION OF THE DRAWINGS

Further advantages, features and embodiments of the invention areindicated in the following examples and the accompanying drawings, whichshow:

FIG. 1 the schematic path of synthesis for the bifunctional vitamin Dderivative 25-hydroxyvitamin-D₃-3β-3′[6-N-(biotinyl)hexamido]amidopropylether) in accordancewith the invention;

FIGS. 2, 3 and 4 schematic representations of various ELISAs for thedetection of 25-OH-vitamin D with the aid of the 25-OH-vitamin Dconjugate in accordance with the invention;

FIG. 5A the calibration curve of a competitive ELISA for 25-OH-vitamin Daccording to FIG. 2;

FIG. 5B calibration curves for ELISAs in accordance with FIG. 5A, having3, 60 and 100 days old 25-OH-vitamin D biotin tracer;

FIG. 5C the calibration curve of a competitive ELISAs for 1,25-dihydroxyvitamin D, analogous to FIG. 2;

FIGS. 6 and 7 schematic representations of competitive RIAs for25-OH-vitamin D with the aid of the 25-OH-vitamin D conjugate inaccordance with the invention;

FIGS. 8, 9 and 10 schematic representations of competitive, radioactiveIRMAs for 25-OH-vitamin D with the aid of the 25-OH-vitamin D conjugatein accordance with the invention;

FIGS. 11 and 12 schematic representations of competitive ELISAs with theemployment of microparticles;

FIG. 13 schematic representations of a competitive binding assay for25-OH-vitamin D with the aid of a 25-OH-vitamin D conjugate inaccordance with the invention and a directly labelled vitamin D bindingprotein;

FIG. 14 a block diagram of the comparison of the 1,25-dihydroxy vitaminD-content in serum from dialysis patients and normal patients.

FIG. 1 shows the route of synthesis in accordance with the invention forthe production of a bifunctional 25-OH vitamin D conjugate. First, 25-OHvitamin D is cyanoethylated in a mixture of acetonitrile, potassiumhydride, and tertiary butanol with acrylonitrile. Due to the presence ofthe potassium hydride, acting as a base, and due to the presence oftertiary butanol for avoiding non-specific reactions at the 25-hydroxygroup, it is achieved that the 3-hydroxy group of the vitamin D isselectively cyanoethylated. The yield of 25-OH vitaminD-3β-cyanoethylether amounts, as a rule, to about 74% with a reactiontime of 40 minutes.

After conventional preparation, the 25-OH vitamin D-3□ cyanoethyletheris mixed with lithium hydride and the 25-hydxoxy group converted intothe lithium alcoholate. There follows a reduction of the nitrite withLiAlH₄, to 25-OH-vitamin-D-3□-3′-amino propylether. This step isquantitative, without by-products arising. Finally there is effected, ifnecessary, a biotinylation with an active biotinylation reagent such asLC-BHNS (biotinyl -N-ε-amino caproyl-hydroxy-succinimide ester). Theresulting spacer group X has, corresponding to the amino caproyl chain,a length of about 0.8 to 0.9 nm.

25-OH-vitamin-D-3β-3[′6-N-(biotinyl)hexamido]amidopropyl-ether istemperature stable and can be stored over many months in an aqueous,slightly acid matrix. Since the compound can not be cleaved by serumenzymes, it is ideally suited for routine diagnostic tests in serum,plasma and urine.

FIG. 2 shows a schematic representation of a competitive ELISA for25-OH-vitamin D. Here, the 25-OH-vitamin D conjugate(25-OH-vitamin-D-3β-3′[6-N-(biotinyl)hexamido]-amidopropylether) isbound via streptavidin to a solid phase. Then, in liquid phase, there iseffected the competitive binding of vitamin D binding protein and25-OH-vitamin D from a standard or a sample to the25-OH-dihydroxyvitamin D conjugate. The detection is effected by meansof peroxidase labelled antibodies against the vitamin D binding protein.The skilled person knows that also other marker enzymes can be employed,for example alkaline phosphatase or galactosidase, etc.

FIG. 3 shows a schematic representation of a competitive,non-radioactive ELISA whereby the vitamin D binding protein is firstbound to the solid phase via anti-vitamin D binding protein antibodies.There is then effected, in liquid phase, a competitive binding of25-OH-vitamin D biotin and 25-OH-vitamin D from a standard or a sample.For detection, peroxidase-labelled streptavidin is then employed. Theindicated principle can of course be transferred to other tracer groupsinstead of biotin and to other marker enzymes, as indicated above.

FIG. 4 shows a schematic representation of a competitive ELISA, wherebythe vitamin D binding protein is directly bound to the solid phase. Thecompetitive binding of 25-OH-vitamin D₃-biotin and 25-OH-vitamin D₃ froma standard or a sample is effected in liquid phase andperoxidase-labelled streptavidin is employed for quantitative detection.

FIGS. 5A-C show the typical calibration curves of competitive ELISAswith 25-OH- or 1,25-dihydroxy vitamin-D₃-biotin, in accordance with theprinciple shown in FIG. 2. The quantity of bound vitamin D bindingprotein was determined by means of a standardised colour reaction withperoxidase-coupled anti-vitamin D binding protein antibodies andtetramethylbenzidine (TMB) as substrate. Alternative substrates wouldbe, for example, OPD (1,2-phenyldiamine×2 HCl), ABTS and others. For thecalibration curve, vitamin D samples with concentrations of 0, 8, 20,50, 125 and 312 nMol/l were employed. The ordinate shows the opticaldensity as the mean value of two measurements at 450 nm; the abscissashows the concentration of 25-OH- or 1,25-dihydroxy vitamin D in nMol/l.

FIG. 6 shows the schematic representation of a competitive proteinbinding test (CPBA), wherein 25-OH-vitamin-D₃-biotin and 25-OH-vitaminD, from a standard or sample, compete in liquid phase for the bindingsite of the vitamin D binding protein. ¹²⁵I-labelled streptavidin isemployed for the quantitative detection.

FIG. 7 shows the schematic representation of a competitiveradioimmunoassay (RIA), wherein 25-OH-vitamin-D-biotin and 25-OH-vitaminD from a standard or a sample compete in liquid phase for the bindingsite of an anti-vitamin D-antibody. ¹²⁵I-labelled streptavidin isemployed for quantitative detection. If the detection is effected bymeans of a streptavidin which is not radioactive but is labelled with afluorophore or luminophore, so-called LIA or FIA assays are involved.

FIG. 8 shows schematic representation of a 25-OH-vitamin D-IRMA. First,25-OH-vitamin-D-biotin is bound to the solid phase via streptavidin. Thecompetitive binding of vitamin D-binding protein to the conjugate and25-OH-vitamin-D₃ from a standard or a sample is then effected in liquidphase. The quantity of the conjugate-bound binding protein is determinedwith ¹²⁵I-labelled antibodies.

FIG. 9 is the schematic representation of an IRMA sandwich technique(immunoradiometric assay). For this purpose, anti-vitamin D₃ antibodiesare coupled to the solid phase. Vitamin D binding proteins then bound tothese. The competition takes place in the next step between the25-OH-vitamin D conjugate and 25-OH-vitamin D from a standard or asample. The determination of the quantity of the bound conjugate iseffected by means of a ¹²⁵I-labelled streptavidin.

FIG. 10 shows the schematic representation of a further S IRMA sandwichtechnique. First, vitamin D₃ binding proteins are coupled to the solidphase. There is then effected thereupon the competitive binding betweenthe 25-OH-vitamin D₃ conjugate and 25-OH-vitamin D₃ from a standard or asample. The quantity of bound conjugate is determined by means of¹²⁵I-labelled streptavidin.

FIG. 11 shows the schematic representation of a competitive ELISAemploying microparticles. Here, 25-OH-vitamin D-biotin is bound tomicroparticles via streptavidin. 25-OH-vitamin D derivative is thenbound thereto. Vitamin D binding protein and the sample concerned arethen added in liquid phase. Binding proteins and 25-OH-vitamin D₃ from astandard or a sample compete for the binding site of the conjugate. Thebound components are separated in that they are held back via themicroparticles by a magnet, whereas the remainder with the non-boundsubstances is removed. The quantity of coupled binding protein isdetermined in a 2-stage process with a primary antibody against vitaminD binding protein and a secondary peroxidase-labelled antibody.

FIG. 12 shows a schematic representation of a competitive ELISAemploying microparticles. 25-OH-vitamin-D-biotin is bound tomicroparticles via streptavidin. Then the liquid sample with25-OH-vitamin D₃ (from a standard or a sample) is added, as is anon-saturating quantity of antibodies. The conjugate and the nativevitamin D₃ compete for the binding of the antibody. The quantity ofbound antibodies is effected by means of agglutination of themicroparticles. This can be determined for example directly by means ofnephelometric analysis or turbimetric analysis.

FIG. 13 shows the scheme of a competitive binding assay, whereby thevitamin D binding protein is directly labelled, for exampleradioactively with ¹²⁵Iodine, or for an electrochemoluminescence, withruthenium(II)tris-(bipyridine)-NHS-ester. The marking may also beenzymes such as peroxidase, alkaline phosphatase, β-galactosidase, etc.,or may also be FITC.

FIG. 14 illustrates in a block diagram the different 1,25-dihydroxyvitamin D contents in serum from dialysis patients and from normalpatients.

The known detection methods for proteins such as the competitive ELISAare based on the principle that the compound to be detected competeswith a binding protein or conjugate for a binding site. Then, thequantity of bound binding protein or conjugate is determined and on thebasis of a calibration curve the concentration of the compound to bedetected is determined.

The test principles shown in the Figures can be carried over simply toother vitamin D derivatives. 1α,25-dihydroxy vitamin D₂ and D₃ are to beparticularly mentioned. In this case a binding protein or a receptor orantibody must be selected which specifically recognises the1α,25-dihydroxy vitamin D analog. The associated bifunctional1α,25-dihydroxy derivative can be obtained enzymatically by means ofreaction of 25-OH-vitamin D-3β-cyanoethylether with25-OH-vitamin-D-1α-hydroxylase, reduction to the amine and finally theaddition of the second functional group. Further, derivatives of vitaminD₂ and vitamin D₃ are here proposed. The synthesis thereof can beeffected through the route set out in Example 1.

EXAMPLES Example 1 Synthesis of25-OH-Vit.-D₃-3β-3′[6-N-(biotinyl)-hexamido]amidopropylether (4)

All reactions were performed in the dark in a dry nitrogen atmosphere.Intermediate products were stored at −20° C. HPLC-pure solvents wereemployed. The 25-OH-vitamin D₃ was obtained from BIOMOL FeinchemikalienGmbH, Hamburg, the LC-BHNS (Long-Chain-Biotinyl-N-ε-aminocaproyl-hydroxy-succinimide ester) from Sigma Chemie, and all furtherchemicals from Fluka, Darmstadt. The mass spectroscopy (FAB) was carriedout with a Finigan-MAT-90, the NMR-measurements with a Bruker-ARX-400(400 MHz) or a Bruker-ARC-250F (250 MHz).

(i) 25-OH-Vitamin D₃-3β-cyanoethylether (2)

5 mg 25-OH-vitamin D₃ (12.5 μMol), dissolved in methylene chloride(CH₂C₂), was transferred into a vial filled with nitrogen and thesolvent was distilled off. The solid remainder was dissolved in 1 mlacetonitrile and mixed with 10 drops of a mixture of tertiary butanoland acetonitrile (9:1 v/v) and 130 μMol acrylonitrile (10 eq.) in 100 μlacetonitrile [stock solution: 86 μl acrylonitrile (1.3 mMol) dilutedwith acetonitrile to 1 ml]. The clear solution was stirred for 15minutes at 6° C. 6.25 μMol potassium hydride (0.5 eq.) in 25 μl tertiarybutanol/acetonitrile (9:1 v/v) [stock solution: 10 mg KH (250 μMol) in 1ml tertiary butanol/acetonitrile (9:1 v/v)] was added. The flocculationthereby arising dissolved again immediately. The mixture was stirred at6° C. Repeated thin layer chromatography (DC) of individual samples with20% petrolether in methyl-tert.-butylether (MTBE) on silica gel showedthat after 10 minutes 90% of the initial compound had been reacted.After 15 minutes a few drops of the reaction mixture were prepared withabout 5 drops of water and 0.5 ml MTBE. The thin film chromatography ofthe organic phase showed no further educt. After 40 minutes the entirereaction mixture was prepared with water/MTBE. 4 mg oleaginous productwas obtained.

IR (NaCl/CH₂Cl₂): 3422 OH 2941, 2872 CH 2252 nitrile 1105 ether

The HPLC-analysis (3% MeOH/CH₂Cl₂) showed 93% product and 7% educt.Thus, 4 mg product contained 3.7 mg (8.2 μMol) target compound, whichcorresponds to a yield of 74%.

(ii) 25-OH-vitamin D₃-3β-3′amino propylether (3)

3.75 mg (8.3 1Mol) nitrile from (i) was dissolved in 2 ml ether, towhich was added 125 μMol lithium hydride dissolved in 1 ml ether (stocksolution: 7 mg fresh finely powdered LiH in 7 ml ether) and stirred for1 hour at room temperature in a nitrogen atmosphere. 169 μMol LiAlH₄ wasadded as suspension in 1 ml ether (base: 18 mg fresh finely powderedLiAlH₄ in 3 ml ether). After a further hour the mixture was preparedwith 1 ml concentrated KOH, 5 ml H₂O and 4×20 ml MTBE. The thin filmchromatography of a sample with 1:1 MTBE/petrolether on silica gelshowed only the starting point. The diole was at R_(i) 0.27; the nitrileat R_(i) 0.4. The substance obtained was processed further withoutfurther analysis and purification.

(iii) 25-Hydroxy vitaminD₃-3β-3′[6-N-(biotinyl)hexaamido]-amidopropylether (4)

3 mg (6.6 μMol) ²5-OH-vitamin D₃-3β-amino propylether (3) from (ii) wasdissolved in 1 ml dimethylformamide (DMF). Then, in a nitrogenatmosphere, 3 mg (6.6 μMol) LC-BNHS and 1 μl (17.5 μmol) triethylaminewere added. Stirring for 18 hours at room temperature took place, theDMF was distilled off and the residue pre-purified with 20% methanol(MeOH) in CH₂Cl₂. 12 mg (>100%) of the substance so obtained waspurified by means of HPLC (conditions: Knauer Kromasil-100, 5 μM, 250×4mm, 10% MeOH in CH₂Cl₂, 1.5 ml/min, OD 265 nm, 7 minutes). The yieldamounted 1.2 mg (1.5 μMol). This corresponds to 129 referred to the25-OH-vitamin D₃ and 18% referred to the nitrile compound.

TABLE I Biotin-25-OH-Vitamin D₃ Cc H Mult [Hz] Assignment 6.42 1 Dd 5.7NH (Biotin) 6.2 1 D 11  6 6.0 1 D 11  7 5.85 1 Dd 5.7 NH (Biotin) 5.55 2M 3-O—CH₂(28) 5.38 1 S NH or OH 5.05 1 D 2 19 4.83 1 D 2 19 4.77 1 S NHor OH 4.51 1 M HC—NH I Biotin 4.33 1 M HC—NH II Biotin 3.53 1 M  3 2.531 D 10  4 1.21 6 S 26,27-CH₃ 0.93 3 D 6 21-CH₃ 0.54 3 S 18-CH₃

MS (Finigan MAT 90); (FAB): 797 (MH⁺) of 5.9.97 and 28.11.97; ¹H-NMR(Bruker ARX 400) in CDCl₃/TMS at 400 MHz. The data of the analysis areshown in table I.

Example 2 Stability of25-OH-Vit.-D₃-3β-3′[6-N-(biotinyl)-hexamido]amidopropylether

In each case 20 mg purified 25-OH-D₃-biotin compound (25-OH-vitaminD₃-3β-3′[6-N-(biotinyl)-hexamido]amidopropyl-ether) from Example 1 wasplaced in an NMR test tube to which 1 ml solvent was added. The solventwas a mixture of deuterium chloroform:deuterium acetonitrile: D₂O in theratio 3:2:1 with a pH-value between 4 and 5. The samples were stored for200 days under the conditions set out below and the NMR spectra wereinvestigated at regular intervals.

Sample 1: light excluded at −20° C.;

Sample 2 light excluded at +4-6° C.;

Sample 3: light excluded at room temperature;

Sample 4: subject to strong light (on a window ledge) at roomtemperature.

Samples 1 and 2 showed no substantial alteration in NMR-spectrum overthe entire time. An HPLC analysis confirmed that samples 1 and 2 wereintact even after 200 days in protonic solvent. Sample 3 showed aminimal alteration of NMR spectrum after 100 days. The HPLC analysisindicated that more than 78% of the compound was still intact. Sample 4was degraded after two months. The investigation of stability shows thatthe compound is very stable when light is excluded, even in protonicsolvent and without cooling.

Example 3 25-Hydroxy Vitamin D-ELISA with 25-Hydroxy VitaminD₃-3β-3′[6-N-(biotinyl)-hexamido]amidopropylether

The detection was effected in accordance with the principle illustratedin FIG. 2. For this purpose, 25-OH-vitaminD-3β-3′[6-N-(biotinyl)hexamidol]amidopropylether had to be bound to asolid phase via streptavidin.

(i) Coating a Microtitration Plate with Streptavidin

Into each of the wells of a microtitration plate there was placed 100 ngstreptavidin, dissolved in 200 μl 60 nM NaHCO₃, pH 9.6, and the plateincubated overnight at 4° C. The streptavidin solution in the well wasremoved and each well washed five times with 200 μl washing buffer (PBS,pH 7.4 with 0.05% Tween-20). Then, 250 μl assay buffer was placed ineach well. For the assay buffer, 5 g casein was dissolved in 100 ml 0.1N NaOH and topped up with PBS, pH 7.4 to 1 L volume. The solution wasboiled for one hour, the volume supplemented to 1 liter with distilledwater, the pH value set to 7.4 and 0.1 g thimerosal added to avoidgrowth of microbes. The wells in the microtitration plate were incubatedfor 1 hour at room temperature with assay buffer, then the assay bufferwas removed and each well washed five times with in each case 200 μlwashing buffer.

(ii) Binding of 25-Hydroxy vitaminD₃-3β-3′[6-N-(biotinyl)-hexamido]amidopropylether

Into each well there was introduced 100 μl biotin-vitamin D-solution (10ng 25-OH-vitamin D-3β-3′[6-N-(biotinyl)hexamido]amidopropylether in 100μl washing buffer) and incubated for one hour at room temperature, inthe dark whilst being shaken. Then, the biotin-vitamin D-solution wasremoved from the wells and each well washed five times in each case with200 μl washing buffer. In the liquid phase, there was effected acompetitive binding of vitamin D binding protein in the presence of25-OH-vitamin D from a standard or a sample.

(iii) Sample Preparation

50 μl serum was mixed by vortexing with 200 μl ethanol_(abs) (pre-cooledto −20° C.) in a 1.5 ml Eppendorf reaction vessel and precipitated for20 minutes at −20° C. The samples were centrifuged at maximum speed ofrotation in an Eppendorf table centrifuge and the result removed andplaced in the ELISA.

One can as a rule assume that plasma or serum samples are stable forabout two weeks at 4° C. In the case of longer storage they must be deepfrozen until they are analysed. Before storage, urine samples must beset to a pH-value between 6 and 8 with 1 N NaOH. Then, they may bestored at 4° C. for about 14 days; in the case of longer storage thesealso must be deep frozen until the analysis is carried out.

(iv) Competitive Binding

In each case 100 μl vitamin D binding protein, isolated from goat serum(1:15000 in assay buffer with 3% (w/v) PEG 6000) together with 10 μlstandard, control or sample (10 μl result from the sample preparation)was placed in the wells. The microtitration plate was incubated for 24hours at 4° C. in the dark and subject to shaking. Then, the solutionwas removed from the wells and the wells washed five times in each casewith 200 μl washing buffer.

(v) Detection of the Competitive Binding

In each case 100 μl rabbit-anti-vitamin D-binding-protein (1:10000diluted in assay buffer having 3% (w/v) PEG 6000) was introduced intothe wells and incubated for 1 hour in the dark and subject to shaking,at room temperature. The solutions were removed from the wells and eachwell washed five times with in each case 200 μl washing buffer. Thequantitative determination was effected with 100 μlanti-rabbit-IgG-peroxidase (1:20000 diluted in washing buffer).Incubation took place for 1 hour at room temperature. Thereafterantibody solutions were taken off and each well washed five times ineach case with 200 μl washing buffer. For the colour reaction 100 μltetramethylbenzidine(TMB) substrate solution (ready for use, from NOVUMDiagnostika GmbH, Dietzenbach, Germany) was introduced into the wells.After 30 minutes the colour development was stopped by the addition of50 μl 2 M H₂SO₄ per well. The measurement of the optical density waseffected at 450 nm. The following tables II and III show the pipettingscheme for the microtitration plate and the values for the opticaldensity.

As standards there were employed solutions of 25-OH-vitamin D₃ in assaybuffer with the following concentrations: 0, 8, 20, 50, 125 and 312nMol/L (see calibration curve in FIG. 5A). As controls or samples thereserved four serums from patients having a D-hypovitaminosis (sample nos.24, 203, 963, 965) and four randomly chosen normal serums (sample nos.NP 18, NP 25, NP 34, NP 37—test series 3 and 4). For the vitaminD-deficiency serums additionally the 25-OH-vitamin D concentration wasdetermined by means of competitive binding assay with the aid of³H-25-OH-vitamin D. As a further “controls” there served four solutionsfor which the respective concentrations of 25-OH-vitamin D were knownfrom other determinations, either from manufacturer information or bymeans of a competitive binding assay (CBPA) with ³H-25-OH-vitamin D.

TABLE II Sample arrangement Duplicate Duplicate Duplicate PipettingStandard value of Serum value of value of Scheme nMol/L column 1 sampleNo. column 3 Controls column 5 Row/ 1 2 3 4 5 6 Column A NSB NSB 24 24K1 (CPBA) K1 (CPBA) B 0 0 203 203 K2 (CPBA) K2 (CPBA) C 8 8 963 963 K3(HPLC) K3 (HPLC) D 20 20 965 965 K4 (HPLC) K4 (HPLC) E 50 50 NP 18 NP 18F 125 125 NP 25 NP 25 G 312 312 NP 34 NP 34 H NP 37 NP 37 NSB:Non-specific binding buffer (Assay buffer without Vitamin D bindingprotein)

TABLE III Measurement values after 30 minutes colour developmentDuplicate Duplicate Duplicate value for Serum value for value for OD 450nm Standard column 1 sample No. column 3 Controls column 5 Row/ 1 2 3 45 6 Column A — — 0.947 1.023 1.903 2.300 B 2.256 2.182 0.853 0.910 0.3930.371 C 1.845 1.861 0.646 0.637 1.674 1.586 D 1.432 1.456 1.429 1.3030.578 0.634 E 0.625 0.612 0.524 0.547 F 0.287 0.261 0.454 0.419 G 0.1560.176 0.341 0.368 H — — 0.421 0.386 B_(max) 2.801 2.676

From the mean values of columns 1 and 2 and the known concentration of25-OH-vitamin D, the calibration curve shown in FIG. 5A was produced.The ordinate shows the optical density as mean value of the twomeasurements at 450 nm; the abscissa shows the concentration of25-OH-vitamin D in nMol/l. The results are summarised in Table V.

Example 4 Comparative Binding Analysis with ³H-25-OH-vitamin D asCompetitive Partner

Insofar as no other indication is given, all reagents, buffers andmaterials were the same as in above-mentioned Example 3. There served ascompetitive binding partner (tracer) tritium-labelled 25-OH-vitamin D₃.Differing from Example 3, the measurement samples were purified by meansof extraction (into individual values). For this purpose, in each case50 μl sample [non-specific assay buffer NSB, standard, control, patientssample (plasma, serum or urine)] was introduced into a 1.5 ml disposablereaction container, 200 μl acetonitrile added, mixed, the containerwalls centrifuged free, and the mixture incubated for 20 to 30 minutesat 4° C. The mixture was centrifuged at 1700 ×g for 10 minutes. Thedeterminations were effected with the results using duplicate values.

For this purpose 25 μl clear result was transferred to a glass test tube(or into a special-RIA-container from Sarstedt, Darmstadt) and 10 μltracer (³H-25-OH-D), 300 μl assay buffer and 100 μl vitamin D-bindingprotein (not in NSB) added. The test tube contents were mixed, incubatedfor one hour at 4° C. and, to remove non-bound radioactive tracer, 100μl activated charcoal suspension (activated charcoal containingphosphate buffer with 0.1% NaN₃) was added. The test tube content wasmixed, incubated for 3 to 5 minutes at 4° C., and the active charcoalpelletized by means of centrifuging for 10 minutes at 1700 ×g. Then, ineach case 400 μl of the result was transferred to a counter container (7ml) and, after the addition of 2 ml scintillator liquid such asAquasafe™ 300 or HiSafe™ III, the radioactivity present in the resultwas counted (2 minutes in a beta-counter). The measurement value for thecontrols, after production of the calibration curve, are shown in TableV.

The comparison with the ELISA according to Example 3 shows that for bothassay procedures (ELISA and CBPA) it is the case that the normal rangefor 25-OH-vitamin D in plasma or serum is about 25-125 nmol/l. Thesensitivity limit of the test systems was determined as B⁰+2SD. Itamounts to about 2.5 nmol/l.

Cross reactions: To serum treated with activated charcoal there wasadded 25-OH-vitamin D₂ (125 nmol/l), 24,25-(OH)₂-vitamin D₃(250 nmol/1)and 1,25-(OH)₂-vitamin D₃ (250 nmol/l). The 25-OH-vitamin D₂cross-reacted to 60%, the 24,25-(OH)₂-vitamin D₃ cross-reacted to 100%,whereas the 1,25-(OH)₂-vitamin D, show no cross-reactivity. Similarresults have been found or expected also for multifunctional25-OH-vitamin D conjugate in accordance with the invention.

Reproducibility: In repeat measurements (n=11) of a sample containing25-dihydroxy vitamin D₃ the following results were achieved. Similarapplies also for measurements with the aid of the multifunctional25-OH-vitamin D conjugate in accordance with the invention:

TABLE IV Intra-assay variance: Mean value Variance Number nmol/l %Sample 1 32 11.3 12.5 Sample 2 32 318 7.2 Inter-assay variance: Meanvalue Variance Number nmol/l % Sample 1 9 9.9 17 Sample 2 9 310 11Clinical: Mean value Number nmol/l Normal persons 35 54 Patients havinghip joint fractures 43 9.5

For the samples mentioned in Example 3 the following 25-OH-vitamin Dconcentrations were determined with the methods according to Examples 3and 4.

TABLE V ELISA ELISA with with Alternative Serum 25-OH-D- CBPA with25-OH-D- determina- sample Biotin ³H-25-OH-D Biotin tion No. nMol/LnMol/L Controls nMol/L nMol/L  24 32.9 33.3 K1 Not 20 (a) measured 20336.8 29.19 K2 76.8 75-125 (a) 963 48.9 38.4 K3 15.0 20-33 (b) 965 21.815.8 K4 51.3 72-120 (b) NP 18 57.0 NP 25 67.9 NP 34 82.4 NP 37 72.9 (a)CBPA with ³H-25-OH-D (b) Manufacturer information

The values indicated by the manufacturers were in general higher thatthe concentrations determined in the competitive binding assay. Thissuggests that in the supplied samples a significant part of the25-OH-vitamin D had already decayed or transformed through the action oflight.

Example 5 Checking of the ELISA-determination by Means of HLPC

Thus, for various samples the 25-OH-vitamin D concentration wasdetermined by means of the ELISA according to Example 3 and, for thepurpose of checking, by means of HPLC. For the calibration curve,standards were employed having vitamin D, concentrations of 0, 8, 20,50, 125 and 312 nMol/l. All samples and standards were measured withduplicate values. The 25-OH-vitamin D₃-concentration of the samples wasthen determined on the basis of the calibration curve from the mean ofthe duplicate values.

The results are shown in the following table VI.

TABLE VI 25-OH-Vitamin D₃ (nMol/L) Sample HPLC ELISA 1 20-33  30 272-120 76 3 79-102 96 4 <15 <Sensitivity limit 5 <15 7.4

Example 6 Long Term Stability of 25-hydroxy Vitamin D-conjugate in theELISA Detection

Calibration curves were repeated with the same standard solutions andreagents according to Example 3, after 60 and 100 days, in order todetermine to what extent an ELISA detection using thebiotin-25-OH-vitamin D-conjugate in accordance with the inventionchanged with the passage of time, when the reagents were stored in theinterim at 4 to 6° C. in the dark. The table below shows the respectiveoptical densities after 30 minutes development (see Example 3).

TABLE VII Duplicate After Duplicate After Duplicate value for 60 valuefor 100 value for Standard Standard column 1 Days column 3 Days Column 5nMol/L 1 2 3 4 5 6 NSB — — 0.191 0.280 0.088 0.109  0 2.256 2.182 2.2272.285 1.471 1.562  8 1.845 1.861 2.041 2.125 1.345 1.366 20 1.432 1.4561.860 1.903 1.079 1.060 50 0.625 0.612 1.293 1.214 0.610 0.690 125 0.287 0.261 0.606 0.615 0.442 0.329 312  0.156 0.176 0.448 0.434 0.2930.257

If the values of the various calibration curves, deducting therespective non-specific binding, are presented in a diagram (see FIG.5B) it can readily be seen that the calibration curves have the sameshape apart from a relative vertical displacement. This shows that thesensitivity and specificity of the ELISA test had not changed over theabove-mentioned period of time.

Example 7 25(OH)-vitamin D₃-ELISA-MTP with Anti-vitamin-D-bindingProtein

The trial was effected in substance in accordance with the protocol ofExample 3 and with the principle illustrated in FIG. 4. The followingbuffers were employed: a) washing buffer: PBS, pH 7.4 with 0.05%Tween-20; b) assay buffer: 5 g casein was dissolved in 100 ml 0.1 N NaOHand supplemented with PBS, pH 7.4 to 1 1. Then 3% (w/v) PEG-6000 and 0.1g Thimerosal™ were added. All incubations were effected in the dark andsubject to shaking.

(i) Coating the Microtitration Plate

Into the wells of a microtitration plate there were introduced in eachcase 100 μl rabbit-anti-vitamin D-binding protein in 60 mM NaHCO₃, pH9.6, and the plate incubated overnight at 4° C. The solutions wereremoved and each well washed five times with 200 μl washing buffer.Then, 250 μl assay buffer was introduced into each well and the plateincubated for 1 hour at room temperature. The assay buffer was removedand each well was washed five times with in each case 200 μl washingbuffer.

(ii) Sample Preparation

50 μl serum, plasma or standard was mixed in a 1.5 ml Eppendorf reactioncontainer with 200 μl ethanol^(abs) (pre-cooled to −20° C.), vortexedand then precipitated for 20 minutes at −20° C. The samples werecentrifuged in an Eppendorf table centrifuge at maximum rotations. Theresult was taken and employed in the ELISA.

(iii) ELISA

Firstly, into each individual well 100 μl vitamin D-binding protein,diluted in assay buffer, was introduced and incubated for 1 hour at roomtemperature. The plate was then knocked out and each individual wellwashed five times in each case with 200 μl washing buffer.

Thereafter, there was introduced into the wells in each case 100 μlbiotin-vitamin D, diluted in assay buffer, together with 10 μl standard,sample or control. The plate was incubated for 24 hours at 4° C. Thesolutions were again removed and each well washed five times in eachcase with 200 μl washing buffer.

As a third step there was introduced into the wells in each case 100 μlperoxidase-coupled streptavidin in a 1:10000-dilution in washing buffer,and incubated for 45 minutes at room temperature. The plate was knockedout and each well washed five times in each case with 200 μl washingbuffer.

For the colour reaction, there was introduced into each well 100 μlTMB-substrate solution. After sufficient colour development (30 minutes)the reaction was stopped with 50 μl 2M H₂SO₄ per well. The measurementof the optical density was defected at 450 nm. Similar to same resultswere obtained as in Example 3 or table V.

Example 8 Content of a Test Pack or a Reagent Set for the Detection of25-hydroxy Vitamin D and 1α,25-dihydroxy Vitamin D

Content of the test pack or test reagents and their preparation:

Standards, for example 6 vials of 25-OH-vitamin D standards with theconcentrations 0, 8, 20, 50, 125 and 312 nmol/l; ready for use inwashing buffer.

Microtitration plates, for example coated with streptavidin, sterilepacked and pre-washed.

Buffer solutions, for example washing buffer, NSB-buffer and assaybuffer, stopper solution.

Controls, for example 2 vials 25-OH-vitamin D controls in human serum.Control 1 (30 nmol 25-OH-D/L), control 2 (80 nMol 25-OH-D/L).

Tracer, for example a vial with biotin-vitamin D (25-OH-vitaminD₃-3β-3′[6-N-(biotinyl) -hexamidol]amidopropylether) in washing buffer(100 ng/ml).

Vitamin D-binding protein, for example a vial with binding protein fromgoat serum in phosphate buffer with 0.1% NaN₃ as stabilising agent.

Marker, for example a vial of anti-rabbit-IgG-peroxidase in washingbuffer.

TMB-developer-solution, for example a vial of stabilisedtetramethylbenzidine-developer solution in washing buffer.

Example 9 ELISA for the Quantitative Detection of 1,25-dihydroxy VitaminD

The detection of 1,25-vitamin D₃ was effected in accordance with theprinciple illustrated in FIG. 2, except that 1,25-dihydroxy vitaminD₃-biotin compound served as tracer. In the competition, 1,25-dihydroxyvitamin D₃ from a standard or a sample, together with a 1,25-dihydroxyvitamin D binding protein, a monoclonal mouse-anti-1α,25-dihydroxyvitamin D-antibody (B. Mawer et al. in Steriods, 1985, 46, 741-754),were brought together. The 1,25-dihydroxy vitamin D₃ from a standard ora sample and the immobilised 1,25-dihydroxy vitamin D₃-biotin compoundthen compete for the binding site of the antibody. The detection iseffected by means of peroxidase-labelled antibodies(goat-anti-mouse-IgG-POX).

(i) The coating of the microtitration plate with streptavidin waseffected as in Example 3, whereby however the washing buffer contained0.1% Triton™ X-100 as a detergent. Otherwise than as in example 3, thewells in the microtitration plate were no longer washed with washingbuffer after the treatment with streptavidin solution, but in each casetreated for 1 hour with 250 μl aqueous sorbitol solution (Karion™ F 1:4in water). The binding of the tracer (1,25-dihydroxy vitamin D-biotin)was effected as in Example 3, except that there was introduced into eachwell 200 μl tracer solution (20 ng 1,25-dihydroxy vitaminD₃-3β-3′[6-N-(biotinyl)-hexamido]-amidopropylether in washing buffer).The 1,25-dihydroxy vitamin D-biotin was synthesised as schematicallyillustrated in FIG. 1, except that after the first step the excess3-cyanoethylated 1-OH-vitamin D intermediate compound was isolated.There can however, also be isolated as desired one of the followingintermediate compounds or, after a mixed synthesis, specifically the1,25-dihydroxy vitamin D₃-3β-3′[6-N-(biotinyl)-hexamido]amidopropyletherby means of HPLC.

(ii) Since in human serum the ratio of 25-OH-vitamin D₃ to1,25-dihydroxy vitamin D₃ as rule is in the range of 1000:1 thequantitative detection of 1,25-dihydroxy vitamin D requires a thoroughpreparation of the samples by means of a combined distribution andabsorption chromatography. In the first step, for this purpose,Extrelut™ Kieselguhr columns (Merck, Darmstadt) are brought toequilibrium each with 500 μl tris-buffer and then there is applied tothe columns in each case 500 μl of a standard, control or investigationsample—in duplicates; the samples can then draw into the columns for 10minutes. The separation of the vitamin D-compounds from the Extrelut™columns was effected by means of four times 1 ml diisopropylether atintervals in each case of 3minutes. The Extrelut™ extract was directlytransferred to a silica cartridge (Merck, Darmstadt) and the Extrelut™columns disposed of. The silica columns were washed five times with 2 mlisopropanol/hexane (4/96 v/v) and 3 times with 2 ml isopropanol/hexane(6/94 (v/v)). The 1,25-dihydroxy vitamin D was then eluded from thesilica columns with two times 2 ml isopropanol/hexane (25/75 v/v) anddried in a nitrogen atmosphere at 37° C. or in a vacuum centrifuge. Thestandard and investigation samples were finally taken up in 20 μlethanol p.a., in each case with 200 μl mouse-anti-1,25-dihydroxy vitaminD-antibody solution (1:150000 in RRA assay buffer: 50 mM KH₂PO₄, 15 mMKCl, 1.25 mM EDTA, 3 mM mercaptoethanol, pH 7.5) and pre-incubated for 1hour at room temperature—as far as possible at the same time as theapplication of the 1,25-dihydroxy vitamin D-biotin tracer to thestreptavidin treated microtitration plate.

(iii) The wells of the tracer-coated microtitration plate were washedfive times in each case with 300 μl Triton™ washing buffer and knockedout onto absorptive paper. Then, 200 μl antibody sample solution fromthe pre-incubation was transferred into the wells and incubated for 1hour in the dark and subject to shaking at room temperature. After theremoval of the solutions from the wells they were washed five times ineach case with 200 μl washing buffer. The quantitative determination waseffected analogously to Example 3 by means of 1 hour incubation with 200μl rabbit-anti-mouse-IgG-peroxidase (1:10000 in washing buffer), at roomtemperature, five times washing of the wells with 300 μl washing buffer,a colour reaction in the dark with 200 μl TMB substrate solution (readyfor use from NOVUM Diagnostika GmbH, Dietzenbach) stopping of the colourreaction after 15 minutes by means of the addition of 50 μl 2 M H₂SO₄and determination of the extinction at 450 nm.

The following table VIII shows the results of the 1,25-dihydroxy vitaminD determination in serum from 11 dialysis patients and six randomlychosen normal persons. For determination of the calibration curve or asstandard, there were employed solutions of 1,25-dihydroxy vitamin D inassay buffer with the following concentration: 0, 6.6, 20, 60 and 180pg/ml (see calibration curve in FIG. 5C).

TABLE VIII Standard Pipetting 1,25-OH-Vit. D OD Double Mean Standardscheme (pg/ml) Remarks 450 nm value value deviation 1 0 Calibration0.784 0.781 0.782 0.002 2 6.6 curve - 0.732 0.741 0.737 0.006 3 20 SeeFIG. 5c 0.682 0.628 4 60 Desired range: 23-63 pg/ml 0.484 0.484 5 180Mean:  43.21 pg/ml 0.233 0.233 Control 50.1 S.D.:   6.62 pg/ml 0.493Serum 98-08-295 Sample Measured value Number (pg/ml) 1 6.5 Serum samples0.705 0.733 0.719 0.020 2 39.1 from dialysis patients 0.564 0.508 0.5360.040 3 57.8 Mean value: 20.5 0.475 0.458 0.466 0.012 4 12.2 S.D. 17.20.672 0.687 0.679 0.010 5 0.3 Median 13.4 0.776 0.774 0.775 0.002 6 4.00.667 0.816 0.741 0.105 7 13.4 0.642 0.700 0.671 0.041 8 39.4 0.5650.504 0.535 0.043 9 22.1 0.619 0.618 0.000 10  22.6 0.531 0.700 0.6160.119 11  8.6 0.705 0.705 Comp. samples 1 52.9 Serum sample from 0.5020.464 0.483 0.027 2 42.6 normal persons 0.518 0.525 0.522 0.005 3 35.3Mean value 46.0 0.522 0.583 0.553 0.043 4 32.9 S.D 9.7 0.571 0.556 0.5630.010 5 59.2 Median 47.8 0.410 0.514 0.462 0.073 6 53.1 0.485 0.4800.482 0.003

FIG. 14 illustrates in a bar chart once again the values found fordialysis and normal patients, in accordance with which values the serumof dialysis patients on average contains significantly less active1,25-dihydroxy vitamin D. The great variance of the values for thedialysis patients shows also the need to more closely monitor thecontent of active 1,25-dihydroxy vitamin D in the serum of dialysispatients, in order better to counter the typical consequences of avitamin D deficiency.

What is claimed is:
 1. A method of obtaining a vitamin D compound of theformula:

wherein: R represents a 25-hydroxy side-group of vitamin D₂ or ofvitamin D₃; Y represents hydrogen or hydroxy; A represents a functionalgroup, coupled via a spacer group, which can be bound by a protein withhigh affinity; comprising; a) cyanoethylating the 3-hydroxy group of avitamin D starting compound in the presence of potassium hydride andtertiary butanol; b) adding lithium hydride and converting the25-hydroxy group into the lithium alcoholate and subsequently reducingthe nitrile group with lithium aluminum hydride; and c) linking a spacergroup together with a functional group A on the amino propylether sidechain.
 2. The method according to claim 1, wherein the functional groupA is selected from biotin, digoxigenin, amino acids, characteristicamino acids and peptide sequences, FITC, proteins and peptide groups,protein-A, protein G and vitamin D derivatives.
 3. The method accordingto claim 1, wherein the functional group A is 25-hydroxy vitamin D or1α,25-dihydroxy vitamin D.
 4. The method according to claim 1, whereinthe functional vitamin D group is coupled in the 3β-position via anether bridge with the spacer group.
 5. The method according to claim 1,wherein step c) is effected with biotinyl-n-ε-aminocaproyl-hydroxy-succinimide ester (LC-BHNS) or an activatedbiotinylation reagent.
 6. Method according to claim 1, wherein thespacer group is an amino carboxylic acid radical, an amino undecanoicacid radical or an amino polyether radical.
 7. A method of producing a3-amino propylether-25-hydroxy or 3-amino propylether-1α,25-dihydroxyvitamin D intermediate compound, comprising; a) cyanoethylating the3-hydroxy group of a vitamin D starting compound in the presence ofpotassium hydride and tertiary butanol; b) adding lithium hydride andconverting the 25-hydroxy group into the lithium alcoholate andsubsequently reducing the nitrile group with lithium aluminum hydride.8. A vitamin D compound of the formula:

wherein; R represents a 25-OH side group of vitamin D, or Y representshydrogen or hydroxyl and X represents a substituted or non-substitutedhydrocarbon group of 0.8 to 4.2 nm length, which optionally contains theheteroatoms S, O, N, and P.
 9. The vitamin D compound according to claim8, obtained by a process comprising a) cyanoethylating the 3-hydroxygroup of a vitamin D starting compound in the presence of potassiumhydride and tertiary butanol; b) adding lithium hydride and convertingthe 25-hydroxy group into the lithium alcoholate and subsequentlyreducing the nitrile group with lithium aluminum hydride to generate anamine; and c) linking two amino-vitamin D groups together by condensingwith a dicarboxylic acid.